Figure 1.
Intrathecal (i.t.) injection of ephrinB1-Fc produced a dose- and time-dependent hyperalgesia and spinal Fos protein expression.
Paw withdrawal latency to the radiant heat and paw withdrawal threshold to mechanical stimuli were recorded at 1-h, 2-h, 4-h, 8-h, 24-h, and 48-h after ephrinB1-Fc injection. Spinal Fos protein expression was assayed at 2-h after ephrinB1-Fc injection. (A)Intrathecal injection of 0.1 or 0.5 µg ephrinB1-Fc, not 0.02 µg ephrinB1-Fc or 0.5 µg control Fc, produced thermal hyperalgesia and mechanical allodynia. P<0.05, from 0.5 to 24 h time point, ephrinB1-Fc 0.1 or ephrinB1-Fc 0.5 group compared with saline or Fc group. Inset figures showed that the calculated area under the curve (AUC) (−2–48 h) in paw-withdrawal latency (PWL) and paw-withdrawal threshold (PWT) test was significantly decreased in a dose-dependent manner in ephrinB1-Fc group. *P<0.05, **P<0.01 compared with saline or Fc group. n = 8 mice in each group. (B) Representative immunohistochemical staining and the quantitative data of Fos expression in the spinal cord of mice. Intrathecal injection of ephrinB1-Fc (0.02, 0.1, and 0.5 µg), not saline or Fc (0.5 µg), induced a dose-dependent increase in spinal Fos protein expression in superficial lamine of ipsilateral spinal cord,*** P<0.001 compared with saline group, n = 6 mice in each group. Scale bar = 100 µm.
Figure 2.
Intrathecal(i.t.) injection of ephrinB1-Fc induced a dose- and time-dependent increase of spinal PI3K-p110γ and p-AKT expression.
The expression of spinal PI3K-p110γ and p-AKT was assayed at 0, 0.17-h, 0.5-h, 1-h, 4-h, and 8-h time-points in the time-dependent experiments and at 0.5-h time-point in the dose-dependent experiments after ephrinB1-Fc injection. PI3K inhibitor wormannin (0.4 µg) or LY294002 (5 µg) or DMSO was intrathecally injected at 30 min before i.t. ephrinB1-Fc (0.5 µg),the expression of p-AKT in spinal was assayed at 1 h after ephrinB1-Fc injection in PI3K mediated spinal AKT activation experiment. (A, B) The representative bands (top) for the expression of PI3K-p110γ or p-AKT in spinal at different time points after injection of ephrinB1-Fc (0.5 µg) and the quantitative data (bottom) for the expression of PI3K-p110γ or p-AKT. The fold change for the density of PI3K-p110γ or p-AKT normalized to GAPDH or AKT for each sample, respectively. The fold change of PI3K-p110γ or p-AKT level in 0 time point groups was set at 1 for quantifications. *P<0.05, **P<0.01, compared with 0 time point group, n = 4 mice in each group. (C, D) The representative bands (top) for the expression of PI3K-p110γ or p-AKT in spinal after injection of various dose of ephrinB1-Fc (0.02, 0.1, and 0.5 µg). The quantitative data (bottom) for the expression of PI3K-p110γ or p-AKT. The fold change for the density of PI3K-p110γ or p-AKT normalized to GAPDH or AKT for each sample, respectively. The fold change of PI3K-p110γ or p-AKT level in Fc groups was set at 1 for quantifications. *P<0.05, **P<0.01, compared with Fc group, n = 4 mice in each group.(E) The representative bands (top) and the quantitative data (bottom) for the expression of spinal p-AKT. The fold change for the density of p-AKT normalized to AKT for each sample. The fold change of p-AKT level in DMSO-saline group was set at 1 for quantifications.*P<0.05, compared with DMSO-ephrinB1-Fc group, n = 4 mice in each group.
Figure 3.
Inhibition of PI3K prevented and reversed pain behavior induced by i.t. ephrinB1-Fc.
PI3K inhibitors were injected at 0.5-h before or after ephrinB1-Fc injection. Paw withdrawal latency to the radiant heat and paw withdrawal threshold to mechanical stimuli were recorded at 0.5-h, 1-h, 2-h, 4-h, 8-h, 24-h and 48-h after ephrinB1-Fc (0.5 µg) injection in the pre-treatment experiment and at 0.5-h, 1-h, 2-h, 4-h, and 8-h after PI3K inhibitors in post-treatment experiment. (A,B) Pre-treatment with various dose of wortmannin (0.016, 0.08, and 0.4 µg) or LY294002 (0.2, 1, and 5 µg) prevented ephrinB1-Fc-induced thermal hyperalgesia (Top) and mechanical allodynia (bottom) in a dose-dependent manner. The calculated area under the curve (AUC) (−2–24 h) (inside bar figure) was significantly increased in W0.4-, W0.08-, LY5- and LY1-ephrinB1-Fc group in PWL test, and in W0.4-, LY5- or LY1-ephrinB1-Fc group in PWT test, *P<0.05, **P<0.01 when compared with DMSO-ephrinB1-Fc group, n = 8 mice in each group. (C,D) Post-treatment with wortmannin (0.016, 0.08, and 0.4 µg) or LY294002 (0.2, 1, and 5 µg) dose-dependently reversed the established thermal hyperalgesia (top) and mechanical allodynia (bottom) by ephrinB1-Fc (0.5 µg).Compared with ephrinB1-Fc-DMSO group, the calculated area under the curve (AUC) (−2–8 h) (inside bar figure) was significantly increased in ephrinB1-Fc-W0.4-, -W0.08, –LY5,or –LY1 group in PWL test, and in ephrinB1-Fc-W0.4, -W0.08,or –LY5 group in PWT test, *P<0.05, **P<0.01 compared with ephrinB1-Fc-DMSO group, n = 8 mice in each group.
Figure 4.
Inhibition of PI3K partially inhibited or reversed the increased expression of spinal Fos protein by i.t. ephrinB1-Fc.
PI3K inhibitor wortmannin (0.4 µg) or LY294002 (5 µg) or DMSO was intrathecally injected at 30 min before and after i.t. ephrinB1-Fc (0.5 µg). Spinal Fos protein expression was assayed at 2 h after ephrinB1-Fc injection. (A,C) Representative Fos immunohistochemical staining and (B,D) the quantitative data of Fos expression in the spinal cord of mice in pre-treatment experiments or post-treatment experiments. ###P<0.001, compared with Sal-DMSO or DMSO-Sal group, *P<0.05, compared with DMSO-ephrinB1-Fc or ephrinB1-Fc-DMSO group, n = 6 mice in each group, Scale bar = 100 µm.
Figure 5.
Intrathecal (i.t.) pretreatment with EphB1-Fc inhibited formalin-induced inflammatory pain and spinal Fos protein expression, accompanied by the decreased expression of spinal PI3K-p110γ and phosphorylated AKT (p-AKT).
The amount of time spent licking the injected paw and the number of flinching paw were measured from 0- to 5-min (the first phase) and from 10- to 40-min (the second phase) after formalin injection. EphB1-Fc was injected at 30-min before formalin injection. The expression of spinal Fos protein and spinal PI3K-p110γ and p-AKT was measured at 2-h and 30-min after injection of formalin, respectively. (A) Pretreatment of EphBs receptors blocker EphB1-Fc (0.5 µg) decreased the time of licking paw and the number of flinching paw induced by formalin. *P<0.05 or **P<0.01 compared with saline (Sal)-Formalin group, n = 8 mice in each group. (C) The representative immunohistochemical staining and the quantitative data for the decreased formalin-induced Fos expression by pretreatment with EphB1-Fc (0.5 µg), not saline, in the spinal cord of mice. ** P<0.01 compared with Sal-Formalin group, n = 6 mice in each group, Scale bar = 100 µm. (D) The representative bands and the quantitative data for the decreased expression of spinal PI3K-p110γ and p-AKT by injection of formalin after pretreatment with EphB1-Fc (0.5 µg). The fold change for the density of PI3K-p110γ and p-AKT normalized to GAPDH or AKT for each sample. The fold change of PI3K-p110γ and p-AKT levels in Sal-Sal group was set at 1 for quantifications. *P<0.05 compared with Sal-Sal group, # P<0.05 compared with Sal-Formalin group n = 4 mice in each group.
Figure 6.
Intrathecal (i.t.) pre- or posttreatment with EphB1-Fc inhibited chronic constrictive injury (CCI)-induced neuropathic pain and spinal Fos protein expression, accompanied by the decreased expression of spinal PI3K-p110γ and phosphorylated AKT (p-AKT).
Consecutive intrathecal injection of EphB1-Fc on 1-h before CCI and 1-d and 2-d after CCI in the pre-treatment experiment and Intrathecal injection of a single dose of EphB1-Fc on 5-d after CCI in post-treatment experiment were performed. Paw withdrawal latency to the radiant heat and paw withdrawal threshold to mechanical stimuli were recorded on 1-d, 3-d, 5-d, 7-d after CCI in pre-treatment experiment and on 1-d, 3-d, 0.5-h,1-h,2-h,4-h,8-h after EphB1-Fc injection on 5-d,6-d and 7-d after CCI in post-treatment experiment. The expression of spinal Fos, PI3K-p110γ and p-AKT was assayed on 5-d after CCI in pre-treatment experiment and at 2-h after EphB1-Fc injection on 5-d after CCI in post-treatment experiment.(A) Pretreatment of EphB1-Fc (0.5 µg) prevented CCI-induced thermal hyperalgesia in paw-withdrawal latency (PWL) test and mechanical allodynia in paw-withdrawal threshold (PWT)test. * P<0.05, ** P<0.01, compared with EphB1-Fc-CCI, n = 8 mice in each group. (B) The representative immunohistochemical staining and the quantitative data for the decreased CCI-induced spinal Fos expression by pretreatment with EphB1-Fc. ** P<0.01 compared with saline (Sal)-CCI group, n = 6 mice in each group, Scale bar = 100 µm. (C) The representative bands and the quantitative data for the decreased expression of spinal PI3K-p110γ and p-AKT by CCI after pretreatment with EphB1-Fc. The fold change for the density of PI3K-p110γ and p-AKT normalized to GAPDH or AKT for each sample. The fold change of PI3K-p110γ and p-AKT levels in Sal-Sham group were set at 1 for quantifications. * P<0.05 or ** P<0.01 compared with Sal -Sham group, # P<0.05 compared with Sal-CCI group, n = 4 mice in each group. (D)Posttreatment of EphB1-Fc reversed CCI-induced thermal hyperalgesia and mechanical allodynia. * P<0.05, compared with CCI-EphB1-Fc, n = 8 mice in each group. (E) The reversed CCI-induced spinal Fos expression by posttreatment with EphB1-Fc. ** P<0.01 compared with CCI-Sal group, n = 6 mice in each group. (F) The decreased expression of spinal PI3K-p110γ and p-AKT by CCI after posttreatment with EphB1-Fc. *P<0.05 or ** P<0.01 compared with Sham -Sal group, # P<0.05 compared with CCI-Sal group, n = 4 mice in each group.
Figure 7.
PI3K mediated spinal ERK activation induced by i.t. ephrinB1-Fc.
PI3K inhibitor wortmannin (0.4 µg) or LY294002 (5 µg) or DMSO was intrathecally injected at 30 min before i.t. ephrinB1-Fc (0.5 µg). The expression of spinal p-ERK was assayed at 30 min after i.t. ephrinB1-Fc injection. Pre-treatment with both wortmannin (A) and LY294002 (B) inhibited spinal ERK activation induced by i.t. ephrinB1-Fc. The representative bands (top) and the quantitative data (bottom) for the expression of spinal p-ERK after i.t. ephrinB1-Fc (0.5 µg). The fold change for the density of each p-ERK normalized to ERK for each sample. The fold change of p-ERK level in DMSO-Sal group was set at 1 for quantifications. ##P<0.01, compared with DMSO-Sal group, *P<0.05, **P<0.01 compared with DMSO-ephrinB1-Fc group, n = 6 mice in each group.